The association constants and thermodynamic values for binding of thyroxine (T4) and triiodothyronine (T3) to purified human thyroxine-binding globulin have been determined by equilibrium dialysis. At pH 7.4 and 37 degrees, T4 was bound to TBG at a single binding site with association constant, K equals 9.08 plus or minus 0.62 x 10 to the minus 9th power M. T3 appeared to be bound at two different classes of binding sites with n1 equals 1, K1 equals 3.80 plus or minus 0.22 x 10 to the minus 8th power M. N2 equals 0.44 plus or minus 0.22, K2 equals 0.43 plus or minus 0.38 x 10 to the minus 8th power M. The change in entropy made a much greater contribution to the binding of T4 (Delta S degrees equals plus 16.7 cal deg-1 mole-1) than to the binding of T3 (Delta S degrees equals plus 1.4 cal deg-1 mole-1). Both T4 and T3 were bound maximally in the region of physiological pH, pH 6.8 to 7.7. Radioimmunoassay of TBG in serum from normal individuals gave a value of 1.55 plus or minus 0.41 mg/100 ml (mean plus or minus SD, n equals 12) in males and 1.85 plus or minus 0.13 mg/100 ml (n equals 18) in females. Preliminary investigations of the effect of temperature on transport of T3 in vivo into liver of Rana catesbiana tadpoles indicated that there was inhibition but not a complete block in movement of a tracer dose of T3 into nuclear fraction at 5 degrees centigrade compared to 25 degrees centrigrade.